Target Team: Saul H, Lauren M, Carlos Gio O. Saul: *Target (protein/gene name): 6-phosphogluconate dehydrogenase, decarboxylating, putative Saul:*NCBI Gene # or RefSeq#: PF14_0520 Saul:*Protein ID (NP or XP #) or Wolbachia#:XP_001348694.1 Saul:*Organism (including strain): p. falciparum 3D7 Saul:Etiologic Risk Group (see link below): http://www.who.int/ith/diseases/malaria/en/ Saul:*/Disease Information (sort of like the Intro to your Mini Research Write up): Malaria is caused by the protozoan parasite Plasmodium. Human malaria is caused by four different species of Plasmodium: P. falciparum, P. malariae, P. ovale and P. vivax. Humans occasionally become infected with Plasmodium species that normally infect animals, such as P. knowlesi. As yet, there are no reports of human-mosquitohuman transmission of such “zoonotic” forms of malaria. The malaria parasite is transmitted by female Anopheles mosquitoes, which bite mainly between dusk and dawn. Saul:Link to TDR Targets page (if present): http://tdrtargets.org/targets/view?gene_id=2850 Saul:Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.) http://www.ncbi.nlm.nih.gov/gene/812102 Saul:Essentiality of this protein: biding, catalytic activity, coenzyme biding, NADP biding, oxidoreductase binding Is it a monomer or multimer as biological unit? (make prediction athttp://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): Saul:Complex of proteins?: none Saul:Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
yes https://www.ebi.ac.uk/chembldb/index.php/target/inspect/11938
3 papers http://tdrtargets.org/targets/papers/?tid=11938 Saul:*EC#: 1.1.1.44 Saul:Link to BRENDA EC# page: http://www.brenda-enzymes.org/enzyme.php?ecno=1.1.1.44 Saul:-- Show screenshot of BRENDA enzyme mechanism schematic
Gio: Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): mixture of 66.7mMtris-HC1 (pH 7.8), 0.334mM 6-phosphogluconate, 0.134mM NADP+, and 1.7 m~MgSO4 in a 1-ml volume Gio:-- link to Sigma (or other company) page for assay (see Sigma links below) http://www.sigmaaldrich.com/catalog/product/sigma/b2517?lang=en®ion=US Gio:-- -or link (or citation) to paper that contains assay information http://download.springer.com/static/pdf/728/art%253A10.1007%252FBF00504362.pdf?auth66=1412571262_3d630219b69007a0c2da0e8c8be777c5&ext=.pdf Gio:-- links to assay reagents (substrates) pages. Gio:--- List cost and quantity of substrate reagents, supplier, and catalog #
Sigma $106.00 for 100MG Gio:Structure (PDB or Homology model) Gio:-- PDB # or closest PDB entry if using homology model: 4GWG Gio:-- For Homology Model option: no need for homology model Gio:---- Show pairwise alignment of your BLASTP search in NCBI against the PDB Gio:---- Query Coverage: Gio:---- Max % Identities: Gio:---- % Positives Gio:---- Chain used for homology:
Lauren: Current Inhibitors: N/A(Cofactor: SO4) Lauren:Expression Information (has it been expressed in bacterial cells): Lauren:Purification Method: Lauren:Image of protein (PyMol with features delineated and shown separately): Lauren:*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): MCDIGLIGLAVMGQNLSLNISSKGFKIGVYNRTYERTEETMKRAKEENLVVYGYKTVEELINNLKKPRKV ILLIKAGPAVDENISNILKHFEKGDIIIDGGNEWYINSERRIKLCKEKDVEYLAMGVSGGEAGARYGCSF MPGGSKYAYDCVKEILEKCSAQVGNSPCVTYIGPGSSGNYVKMVHNGIEYGDMQLISESYVIMKHILKYD NQKLSEVFNKWNEGILNSYLIEITANILAKKDDLTNNYLVDMILDIAGAKGTGKWTMLEATERGIPCPTM CAALDARNISVFKELRTKAESNFNKDNILIDPNEDLNDFENDLLNALYCCKIISYTQGLFLLKQVSEEMN WKLNLGEIARIWRGGCIIRAVFLDRIANAYKNNEKLELLFLDNEFSDDIKNKLPSLRKIVLMATKYSIPI PAFSASLAYFQMVTSQNLPLNLVQAQRDYFGSHTYRRTDREGNYHTLW Lauren:*length of your protein in Amino Acids: 468 Lauren:Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 52994.0 Lauren:Molar Extinction coefficient of your protein at 280 nm wavelength: 69385 Lauren:TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
Lauren:*CDS Gene Sequence (paste as text only):
ATGTGTGATATTGGTTTGATAGGTTTGGCTGTCATGGGCCAGAATTTAAGTTTGAATATTTCAAGCAAAGGTTTTA AAATTGGTGTTTATAATAGGACATATGAAAGAACAGAAGAAACAATGAAAAGAGCAAAAGAAGAGAATTTGGTTG TTTATGGTTATAAAACAGTAGAAGAATTAATAAATAATTTGAAAAAACCAAGGAAAGTTATTTTATTAATCAAAGCAG GTCCAGCTGTAGATGAGAATATTAGTAATATATTAAAACATTTTGAAAAAGGAGATATAATAATTGATGGTGGGAAT GAATGGTATATTAATTCAGAAAGAAGAATAAAATTATGTAAAGAAAAAGATGTAGAATATTTAGCTATGGGTGTGAG TGGAGGTGAAGCAGGTGCAAGATATGGTTGTTCATTTATGCCTGGTGGTTCTAAATATGCATATGATTGTGTGAA AGAAATATTAGAAAAATGTTCAGCTCAAGTTGGAAATTCTCCTTGTGTTACTTATATAGGTCCAGGTTCCTCAGGG AATTATGTAAAAATGGTACATAATGGAATAGAATATGGAGATATGCAATTAATATCAGAAAGTTATGTAATTATGAAAC ATATATTAAAATATGATAATCAGAAATTATCAGAAGTTTTTAATAAATGGAATGAAGGTATATTAAATTCTTACTTAATT GAAATTACTGCAAATATTCTTGCAAAAAAAGATGACTTAACAAATAATTATTTAGTTGATATGATATTAGATATTGCTG GAGCAAAAGGTACAGGGAAATGGACTATGCTTGAAGCTACCGAAAGAGGTATCCCTTGTCCAACTATGTGTGCT GCTTTAGATGCACGTAATATAAGTGTTTTTAAAGAATTAAGAACTAAAGCAGAATCTAATTTTAATAAAGATAATATTC TTATTGACCCAAATGAAGATTTAAATGATTTTGAAAATGATTTATTAAATGCTCTTTATTGTTGTAAAATTATTTCATAT ACTCAAGGTCTTTTCCTTTTAAAACAAGTTTCTGAAGAAATGAACTGGAAATTAAATTTGGGAGAAATTGCTAGAA TATGGAGAGGTGGTTGTATTATAAGAGCTGTTTTCTTAGATCGTATAGCAAATGCTTATAAAAATAATGAAAAATTAG AATTATTATTCCTAGATAATGAATTCTCAGATGATATTAAAAATAAATTACCTTCCTTAAGAAAAATTGTTCTTATGGCT ACAAAATATTCCATACCAATTCCTGCCTTCTCAGCTAGTTTAGCATATTTTCAAATGGTAACCTCACAAAATTTACCT CTTAATTTAGTGCAAGCCCAAAGGGATTATTTTGGATCACACACATATAGGAGAACAGATCGTGAAGGAAATTATC ACACCTTGTGGTGA Lauren:*GC% Content for gene: 28.6% Lauren:*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): 1 ATGTGCGACATTGGTCTGATTGGCCTGGCGGTTATGGGTCAGAATCTGTCTCTGAATATC
1381 GAGGGCAACTATCATACCCTGTGGTAA Lauren:*GC% Content for gene (codon optimized): 47.8%
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences): Saul Huerta:
Forward Primer:
5’ TACTTCCAATCCATGTGCGACATTGGTCTGATCGG 3’ 35 bp
Reverse primer:
5’ TATCCACCTTTACTGTTACCACAGGGTGTGGTAGT 3’ 35 bp Lauren McGregor: Forward Primer:
Saul: *Target (protein/gene name): 6-phosphogluconate dehydrogenase, decarboxylating, putative
Saul:*NCBI Gene # or RefSeq#: PF14_0520
Saul:*Protein ID (NP or XP #) or Wolbachia#: XP_001348694.1
Saul:*Organism (including strain): p. falciparum 3D7
Saul:Etiologic Risk Group (see link below):
http://www.who.int/ith/diseases/malaria/en/
Saul:*/Disease Information (sort of like the Intro to your Mini Research Write up):
Malaria is caused by the protozoan parasite Plasmodium. Human malaria is caused by four different species of Plasmodium: P. falciparum, P. malariae, P. ovale and P. vivax. Humans occasionally become infected with Plasmodium species that normally infect animals, such as P. knowlesi. As yet, there are no reports of human-mosquitohuman transmission of such “zoonotic” forms of malaria. The malaria parasite is transmitted by female Anopheles mosquitoes, which bite mainly between dusk and dawn.
Saul:Link to TDR Targets page (if present): http://tdrtargets.org/targets/view?gene_id=2850
Saul:Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)
http://www.ncbi.nlm.nih.gov/gene/812102
Saul:Essentiality of this protein: biding, catalytic activity, coenzyme biding, NADP biding, oxidoreductase binding
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html):
Saul:Complex of proteins?: none
Saul:Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
yes
https://www.ebi.ac.uk/chembldb/index.php/target/inspect/11938
3 papers
http://tdrtargets.org/targets/papers/?tid=11938
Saul:*EC#: 1.1.1.44
Saul:Link to BRENDA EC# page: http://www.brenda-enzymes.org/enzyme.php?ecno=1.1.1.44
Saul:-- Show screenshot of BRENDA enzyme mechanism schematic
Gio: Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): mixture of 66.7mMtris-HC1 (pH 7.8), 0.334mM 6-phosphogluconate, 0.134mM NADP+, and 1.7 m~MgSO4 in a 1-ml volume
Gio:-- link to Sigma (or other company) page for assay (see Sigma links below)
http://www.sigmaaldrich.com/catalog/product/sigma/b2517?lang=en®ion=US
Gio:-- -or link (or citation) to paper that contains assay information
http://download.springer.com/static/pdf/728/art%253A10.1007%252FBF00504362.pdf?auth66=1412571262_3d630219b69007a0c2da0e8c8be777c5&ext=.pdf
Gio:-- links to assay reagents (substrates) pages.
Gio:--- List cost and quantity of substrate reagents, supplier, and catalog #
Sigma $106.00 for 100MG
Gio:Structure (PDB or Homology model)
Gio:-- PDB # or closest PDB entry if using homology model: 4GWG
Gio:-- For Homology Model option: no need for homology model
Gio:---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
Gio:---- Query Coverage:
Gio:---- Max % Identities:
Gio:---- % Positives
Gio:---- Chain used for homology:
Lauren: Current Inhibitors: N/A(Cofactor: SO4)
Lauren:Expression Information (has it been expressed in bacterial cells):
Lauren:Purification Method:
Lauren:Image of protein (PyMol with features delineated and shown separately):
Lauren:*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MCDIGLIGLAVMGQNLSLNISSKGFKIGVYNRTYERTEETMKRAKEENLVVYGYKTVEELINNLKKPRKV
ILLIKAGPAVDENISNILKHFEKGDIIIDGGNEWYINSERRIKLCKEKDVEYLAMGVSGGEAGARYGCSF
MPGGSKYAYDCVKEILEKCSAQVGNSPCVTYIGPGSSGNYVKMVHNGIEYGDMQLISESYVIMKHILKYD
NQKLSEVFNKWNEGILNSYLIEITANILAKKDDLTNNYLVDMILDIAGAKGTGKWTMLEATERGIPCPTM
CAALDARNISVFKELRTKAESNFNKDNILIDPNEDLNDFENDLLNALYCCKIISYTQGLFLLKQVSEEMN
WKLNLGEIARIWRGGCIIRAVFLDRIANAYKNNEKLELLFLDNEFSDDIKNKLPSLRKIVLMATKYSIPI
PAFSASLAYFQMVTSQNLPLNLVQAQRDYFGSHTYRRTDREGNYHTLW
Lauren:*length of your protein in Amino Acids: 468
Lauren:Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 52994.0
Lauren:Molar Extinction coefficient of your protein at 280 nm wavelength: 69385
Lauren:TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
Lauren:*CDS Gene Sequence (paste as text only):
ATGTGTGATATTGGTTTGATAGGTTTGGCTGTCATGGGCCAGAATTTAAGTTTGAATATTTCAAGCAAAGGTTTTA
AAATTGGTGTTTATAATAGGACATATGAAAGAACAGAAGAAACAATGAAAAGAGCAAAAGAAGAGAATTTGGTTG
TTTATGGTTATAAAACAGTAGAAGAATTAATAAATAATTTGAAAAAACCAAGGAAAGTTATTTTATTAATCAAAGCAG
GTCCAGCTGTAGATGAGAATATTAGTAATATATTAAAACATTTTGAAAAAGGAGATATAATAATTGATGGTGGGAAT
GAATGGTATATTAATTCAGAAAGAAGAATAAAATTATGTAAAGAAAAAGATGTAGAATATTTAGCTATGGGTGTGAG
TGGAGGTGAAGCAGGTGCAAGATATGGTTGTTCATTTATGCCTGGTGGTTCTAAATATGCATATGATTGTGTGAA
AGAAATATTAGAAAAATGTTCAGCTCAAGTTGGAAATTCTCCTTGTGTTACTTATATAGGTCCAGGTTCCTCAGGG
AATTATGTAAAAATGGTACATAATGGAATAGAATATGGAGATATGCAATTAATATCAGAAAGTTATGTAATTATGAAAC
ATATATTAAAATATGATAATCAGAAATTATCAGAAGTTTTTAATAAATGGAATGAAGGTATATTAAATTCTTACTTAATT
GAAATTACTGCAAATATTCTTGCAAAAAAAGATGACTTAACAAATAATTATTTAGTTGATATGATATTAGATATTGCTG
GAGCAAAAGGTACAGGGAAATGGACTATGCTTGAAGCTACCGAAAGAGGTATCCCTTGTCCAACTATGTGTGCT
GCTTTAGATGCACGTAATATAAGTGTTTTTAAAGAATTAAGAACTAAAGCAGAATCTAATTTTAATAAAGATAATATTC
TTATTGACCCAAATGAAGATTTAAATGATTTTGAAAATGATTTATTAAATGCTCTTTATTGTTGTAAAATTATTTCATAT
ACTCAAGGTCTTTTCCTTTTAAAACAAGTTTCTGAAGAAATGAACTGGAAATTAAATTTGGGAGAAATTGCTAGAA
TATGGAGAGGTGGTTGTATTATAAGAGCTGTTTTCTTAGATCGTATAGCAAATGCTTATAAAAATAATGAAAAATTAG
AATTATTATTCCTAGATAATGAATTCTCAGATGATATTAAAAATAAATTACCTTCCTTAAGAAAAATTGTTCTTATGGCT
ACAAAATATTCCATACCAATTCCTGCCTTCTCAGCTAGTTTAGCATATTTTCAAATGGTAACCTCACAAAATTTACCT
CTTAATTTAGTGCAAGCCCAAAGGGATTATTTTGGATCACACACATATAGGAGAACAGATCGTGAAGGAAATTATC
ACACCTTGTGGTGA
Lauren:*GC% Content for gene: 28.6%
Lauren:*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
1 ATGTGCGACATTGGTCTGATTGGCCTGGCGGTTATGGGTCAGAATCTGTCTCTGAATATC
61 TCCTCTAAGGGTTTCAAAATCGGTGTTTACAACCGTACCTACGAACGTACGGAAGAAACC
121 ATGAAACGTGCGAAAGAAGAGAACCTCGTGGTCTACGGTTACAAAACCGTTGAGGAACTG
181 ATCAATAATCTGAAAAAACCACGTAAGGTTATCCTCCTCATCAAGGCGGGTCCGGCGGTT
241 GACGAAAACATTTCTAATATTCTGAAACACTTCGAGAAAGGTGACATTATCATCGACGGT
301 GGTAACGAATGGTACATCAACTCTGAACGTCGTATCAAACTGTGCAAGGAAAAAGACGTT
361 GAATACCTGGCGATGGGTGTTTCTGGTGGTGAAGCCGGTGCGCGTTACGGCTGTTCTTTC
421 ATGCCAGGTGGCTCTAAATATGCGTACGACTGCGTTAAGGAAATCCTGGAAAAGTGCTCT
481 GCTCAGGTGGGTAACTCTCCATGTGTCACTTACATCGGTCCGGGTAGCTCTGGTAACTAC
541 GTTAAAATGGTTCACAACGGTATCGAATACGGCGACATGCAGCTGATCTCCGAATCTTAC
601 GTCATCATGAAGCACATCCTCAAATACGACAACCAGAAGCTGTCTGAAGTCTTCAACAAG
661 TGGAACGAAGGTATCCTGAATAGCTACCTGATTGAAATCACCGCGAACATCCTGGCAAAG
721 AAAGACGATCTGACCAACAACTACCTGGTTGACATGATTCTCGACATCGCGGGTGCTAAA
781 GGTACCGGCAAATGGACCATGCTGGAAGCGACCGAACGTGGTATCCCGTGCCCGACTATG
841 TGTGCGGCGCTGGATGCCCGCAATATTTCTGTTTTCAAAGAACTCCGTACCAAAGCGGAA
901 TCTAACTTCAATAAAGACAACATTCTCATTGACCCTAACGAAGACCTGAACGACTTCGAA
961 AACGACCTGCTCAACGCGCTCTACTGCTGCAAAATCATCTCTTACACCCAAGGTCTCTTT
1021 CTGCTGAAGCAAGTTTCTGAAGAAATGAACTGGAAACTCAACCTGGGTGAGATCGCGCGT
1081 ATCTGGCGTGGTGGTTGCATCATCCGTGCGGTTTTCCTGGACCGTATTGCGAATGCCTAT
1141 AAGAATAACGAAAAGCTGGAGCTGCTGTTCCTCGACAATGAATTTTCTGACGACATCAAA
1201 AACAAACTGCCGTCTCTGCGCAAAATTGTTCTGATGGCGACCAAATACTCTATCCCGATC
1261 CCGGCGTTCTCTGCTTCTCTCGCGTACTTCCAGATGGTTACCTCTCAGAACCTGCCGCTC
1321 AATCTGGTTCAGGCGCAGCGTGACTACTTCGGTTCTCACACGTATCGTCGTACTGATCGT
1381 GAGGGCAACTATCATACCCTGTGGTAA
Lauren:*GC% Content for gene (codon optimized): 47.8%
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
Saul Huerta:
Forward Primer:
5’ TACTTCCAATCCATGTGCGACATTGGTCTGATCGG 3’ 35 bp
Reverse primer:
5’ TATCCACCTTTACTGTTACCACAGGGTGTGGTAGT 3’ 35 bp
Lauren McGregor:
Forward Primer:
TACTTCCAATCCATGTGCGACATTGGTCTGAT
Reverse Primer:
ACTATCATACCCTGTGGTAACAGTAAAGGTGGATA